Previous studies revealed that an exon encoding 8 amino acids is alternatively spliced into loop 1 of nonmuscle myosin heavy chain (NMHC) II-C, resulting in NMHC II-C1 which is expressed in a number of different tissues. The inclusion of this alternatively spliced exon results in a heavy meromyosin (HMM) with a higher actin-activated MgATPase activity and in vitro motility than non-inserted HMM II-C0. Here, we report on a second alternatively spliced exon encoding 41 amino acids which are incorporated into loop 2 of NMHC II-C (NMHC II-C2) and which are only expressed in neuronal tissues. Unlike all other nonmuscle myosin II isoforms or smooth muscle myosins that require phosphorylation of the 20kDa myosin light chain for actin-activated MgATPase activity and for translocation of actin filaments, baculovirus expressed HMM II-C2 does not require regulatory light chain phosphorylation for full activity. HMM II-C2 has the following kinetic parameters: V10=0.08s-1 (activity at 10uM actin), Vmax=0.12s-1, KATPase=3.88uM in the absence of myosin light chain phosphorylation by myosin light chain kinase (MLCK) and V10=0.12s-1, Vmax=0.13s-1, KATPase=1.36uM following light chain phosphorylation. Addition of the C1 insert into loop 1 of HMM II-C2 (HMM II-C1C2) increases the actin-activated MgATPase activity of HMM II-C2. HMM II-C1C2 has the kinetic parameters: V10=0.12s-1, Vmax=0.32s-1, KATPase=16.23uM in the absence of myosin light chain phosphorylation by MLCK and V10=0.16s-1, Vmax=0.19s-1, KATPase=2.39uM following phosphorylation of the light chain. Both HMM II-C2 and HMM II-C1C2 can translocate actin filaments at a rate 0.07um/sec and 0.06um/sec, respectively, in the absence of myosin light chain phosphorylation, and at a rate 0.06um/sec following light chain phosphorylation. Experiments to understand the function of nonmuscle myosin II-C2 in the context of a living cell are being undertaken.